The Effect And Mechanism Of Apatinib In Killing Non-small Cell Lung Cancer With LKB1 Deficiency By Activating AMPK

Background and object:Tumor suppressor gene Serine Threonine Kinase 11（STK11）,also named as Liver Kinase B1（LKB1）,is a highly conserved serine/threonine kinase.LKB1 is a key molecule in the regulation of cellular substance and energy metabolism.LKB1 regulates the phosphorylation state of downstream AMP-activated protein kinase（AMPK）and AMPK family proteins through its kinase activity,to maintain the normal metabolism of cells,and inhibits the occurrence and progression of tumors.The incidence of LKB1 mutation is about20%in non-small cell lung cancer（NSCLC）.NSCLC with LKB1 deficiency is characterized by enhanced invasiveness,immunosuppressive tumor microenvironment and low expression of PD-L1,and is closely related to poor prognosis of patients.Therefore,there is an urgent need to develop effective therapeutic treatments for NSCLC with LKB1 deficiency to improve the prognosis of these patients.Recently,a phase Ib/II clinical study（NCT04203485）found that the combination of apatinib,a small molecular vascular endothelial growth factor-tyrosine kinase inhibitor（VEGFR-TKI）,with immune check inhibitor（ICI）can significantly improve the survival outcome of patients with advanced NSCLC.More importantly,LKB1-mutant subgroup benefits more to this regimen compared with LKB1 wild type（WT）subgroup,but the mechanism stays unclear.The purpose of this study is to explore the potential mechanism of apatinib in the treatment of NSCLC with LKB1 deficiency,clarify the clinical effects of various VEGFR-TKIs,and thus propose a possible treatment for this subtype of lung cancer.Materials and Methods1.Three LKB1-null NSCLC cell lines were successfully transfected with LKB1re-expression lentivirus,thus constructing LKB1-WT NSCLC cell lines.Proteomics and phosphorylated proteomics analysis were utilized to screened out the potential intracellular target of apatinib in NSCLC with LKB1 deficiency.2.Western blotting（WB）experiments validated that apatinib can activate AMPK in LKB1-null cell lines.The interaction between apatinib or anlotinib and AMPK protein was verified by surface plasmon resonance（SPR）assay.3.Six VEGFR-TKIs commonly used in clinic were used to detected the distinct impact on AMPK by WB experiments.Different impact of 26 VEGFR-TKIs on AMPK were detected by homogeneous time resolved fluorescence（HTRF）assay.Virtual molecular docking was used to detect the binding mode of apatinib or anlotinib to AMPK.4.Bioinformatics analysis and cellular proliferation and toxicity experiments were applied to analyze the fatty acid metabolism disorder in LKB1-null NSCLC cell lines.5.¹³C₆ isotope glucose labeling metabolic flux experiment was used to confirm the inhibitory effect of apatinib on cellular de novo fatty acids synthesis.6.CCK8 cellular proliferation and toxicity experiments,clone formation experiments and apoptosis flow cytometry were used to validate the killing effect of apatinib on LKB1-null NSCLC cell lines.7.The clinical record of four patients with advanced NSCLC were analyzed retrospectively.All four patients carried with STK11 deficiency and had received apatinib monotherapy or combination therapy as posterior line therapy in our institution.Results:1.Apatinib significantly increased the phosphorylation of AMPKα1 Ser496 in LKB1-null cells.Apatinib can directly interact with and activate AMPK,and the binding force of apatinib to AMPK is close to that of A-769662,a direct AMPK activator.2.VEGFR-TKIs commonly used in clinic have distinct impacts on AMPK.Apatinib and anlotinib can directly activate AMPK,axitinib and sunitinib can directly inhibit AMPK,while cabozantinib and lenvatinib have no effect on AMPK.3.Fatty acid metabolism-related pathways,such as fatty acidβ-oxidation,fatty acid metabolism and fatty acid biosynthesis,were significantly activated in LKB1-mutant NSCLC.Cellular experiments showed that limitation of exogenous fatty acid intake or fatty acid synthesis inhibitor alone can not effectively killing LKB1-null NSCLC cells.4.The phosphorylation of acetyl-Co A carboxylase（ACC）induced by apatinib can significantly reduce the proportion of de novo synthetic fatty acids and inhibit the proliferation and survival of tumor cells.5.Under the condition of delipidated culture,apatinib can effectively inhibit the growth and proliferation,and can obviously induced the apoptosis of LKB1-null cells.6.Tumor marker levels and local tumor imaging remained stable for extended periods of time in three patients during receiving apatinib.Apatinib monotherapy or combination therapy significantly improved Progression-free survival（PFS）in three patients and Overall survival（OS）in one patient.The combination of apatinib and gefitinib improved primary resistance to gefitinib in a patient with STK11/EGFR co-mutation.Conclusion:1.In this study,AMPK was identified as the intracellular target of apatinib in LKB1-mutant NSCLC,and apatinib could directly interact with and activate AMPK2.VEGFR-TKIs commonly used in clinic have distinct impacts on AMPK.Apatinib and anlotinib can directly activate AMPK,while axitinib and sunitinib can directly inhibit AMPK.3.There is obvious fatty acid metabolism disorder in LKB1-mutant NSCLC.Fatty acid deprivation combined with apatinib may be an effective treatment strategy for NSCLC with LKB1 deficiency.4.Apatinib can improve the survival outcome of patients with LKB1-mutant NSCLC.